CLONING OF THE RAT PROGESTERONE-RECEPTOR GENE 5'-REGION AND IDENTIFICATION OF 2 FUNCTIONALLY DISTINCT PROMOTERS

To examine some of the molecular mechanisms controlling transcription of the rat progesterone receptor (PR) gene, we have cloned and sequenc ed the 5'-region of the gene. Northern blot analyses with a series of probes identified two regions where distinct subsets of the multiple P R gene transcripts initiated, suggesting the presence of two promoters in the gene. Promoter activities for two gene fragments encompassing these regions, -131/+65 (P; distal) and +461/+675 (P'; proximal), were demonstrated in transient transfection experiments using reporter con structs containing the gene fragments linked individually upstream of the chloramphenicol acetyltransferase (CAT) gene. Cotransfection of P- CAT or P'-CAT constructs containing two upstream GAL4 binding sites in to primary cultures of rat uterine cells with a vector expressing a GA L4 DNA binding domain-VP16 activating region fusion protein resulted i n a 10-fold increase in CAT activity relative to cells transfected wit h either reporter and a vector expressing only the GAL4 DNA binding do main. The estrogen inducibility of the promoter-CAT constructs was ass essed by transfection into MCF-7 breast cancer cells, which contain hi gh levels of estrogen receptor (ER). P'-CAT, but not P-CAT, was induce d by estradiol (E(2); 8-fold). In primary rat uterine cells, which con tain lower levels of ER, P'-CAT required the addition of one upstream consensus estrogen response element (ERE) to be estrogen inducible, wh ereas P-CAT required the addition of two EREs. Point and deletion muta nts of the proximal promoter region in the P'-CAT reporter, screened i n MCF-7 cells, were used to identify a 20-base pair fragment (+617/+63 6) that retained the promoter activity and 50% of the estrogen inducib ility of P'. This fragment contained an ERE-like sequence conserved in 8 of 10 positions relative to the consensus ERE. Two copies of this s equence conferred estrogen inducibility (4-fold) when placed upstream of the distal promoter in P-CAT. To examine PR-dependent stimulation o f the two PR gene promoters by cAMP, P-CAT and P'-CAT reporter constru cts containing two upstream consensus EREs were cotransfected into ER- negative 3T3 cells with an ER expression vector. Induction by E(2) was greater than se-fold for both constructs. Treatment of the cells with agents that increase intracellular cAMP levels, namely cholera toxin plus isobutyl methylxanthine, resulted in CAT activity that was 8% and 51% of the E(2)-stimulated activity for the P and P' constructs, resp ectively. These effects required the presence of the ER expression vec tor, as well as the upstream EREs, and were blocked by treatment with the antiestrogen ICI 164,384. These studies have identified two distin ct promoters in the rat PR gene which exhibit differential responsiven ess to E(2) and to PR-dependent stimulation by cAMP. The functional di fferences between these promoters may lead to altered expression of th e PR A and B isoforms and thereby influence cellular responsiveness to progestins.